Enhancement of l-Pipecolic Acid Production by Dynamic Control of Substrates and Multiple Copies of the pipA Gene in the Escherichia coli Genome.

l-Pipecolic acid is an important rigid cyclic nonprotein amino acid, which is obtained through the conversion of l-lysine catalyzed by l-lysine cyclodeaminase (LCD). To directly produce l-pipecolic acid from glucose by microbial fermentation, in this study, a recombinant Escherichia coli strain with high efficiency of l-pipecolic acid production was constructed. This study involves the dynamic regulation of the substrate concentration and the expression level of the l-lysine cyclodeaminase-coding gene pipA. In terms of substrate concentration, we adopted the l-lysine riboswitch to dynamically regulate the expression of lysP and lysO genes. As a result, the l-pipecolic acid yield was increased about 1.8-fold as compared with the control. In addition, we used chemically inducible chromosomal evolution (CIChE) to realize the presence of multiple copies of the pipA gene on the genome. The resultant E. coli strain XQ-11-4 produced 61 ± 3.4 g/L l-pipecolic acid with a productivity of 1.02 ± 0.06 g/(L·h) and a glucose conversion efficiency (α) of 29.6% in fermentation. This is the first report that discovered multiple copies of pipA gene expression on the genome that improves the efficiency of l-pipecolic acid production in an l-lysine high-producing strain, and these results give us new insight for constructing the other valuable biochemicals derived from l-lysine.

A pipecolic acid-rich branched cyclic depsipeptide ulleungamide C from a Streptomyces species induces G0/G1 cell cycle arrest in promyelocytic leukemia cells.

In this study, screening by LC-MS and cytotoxicity-guided isolation led to the identification of ulleungamide C (1), a previously unknown pipecolic acid-rich branched cyclic depsipeptide, from a soil actinobacterium Streptomyces sp. KCB13F003. The structure of 1 was determined by interpretation of spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Antiproliferative assays using mammalian cancerous cells revealed that 1 inhibits the proliferation of HL-60 human promyelocytic leukemia cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with 1. Results of immunoblotting assays revealed that 1 reduced the expression levels of cyclin-dependent kinase 4 (CDK4), CDK6, retinoblastoma protein (Rb), and phosphorylated Rb, whereas it induced cyclin-dependent kinase inhibitor 1B (p27/Kip1) expression.

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted l-Pipecolic Acids.

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5'-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,ß-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio.

Enzymatic hydroxylation of L-pipecolic acid by L-proline cis-4-hydroxylases and isomers separation.

OBJECTIVES: Establish a complete and efficient method for the preparation of cis-5-hydroxy-L-pipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. RESULTS: For non-heme Fe(II)/α-KG-dependent dioxygenases, α-ketoglutarate (α-KG) has great influence on the stability of Fe(II) ions, which is also the basic of the hydroxylation reaction to the substrate. L-pipecolic acids (L-Pip) was converted to cis-5HPA by whole-cell catalysis in water, which can reduce the loss of Fe(II) ions. 120 mM L-Pip can be transformed to 93% via cell and Fe(II) ions continuous supplementation under the reaction system optimization (the molar ratio of ascorbic acid/FeSO4·7H2O and α-KG/L-Pip were 8:1 and 1:1, respectively). After the catalytic reaction, the amino protection strategy was adopted to improve the resolution of isomer products on silica gel chromatography, and the amino protected cis-5HPA was obtained with a yield of 86.7%. CONCLUSIONS: We established a method which is promising to be used for cis-5HPA largescale preparation. It also provides a suitable reference for this type of enzyme-catalyzed reaction and the hydroxy pipecolic acid isomers separation.

Probing the B- & C-rings of the antimalarial tetrahydro-ß-carboline MMV008138 for steric and conformational constraints.

The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C-C Bond Formation.

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of l-amino acids at α, ß, and γ positions. These enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S,6S)-6-methyl pipecolate 1, from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs γ-elimination of O-acetyl-l-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cγ-Cδ bond in 3 and complete the γ-substitution reaction. CndF displays promiscuity toward different ß-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-ß-keto esters led to the biosynthesis of 6-substituted l-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Total Synthesis of the Proposed Structure of Penasulfate A: l-Arabinose as a Source of Chirality.

The total synthesis of putative penasulfate A was effectively achieved by a convergent strategy with a longest linear sequence of 14 steps and overall yield of 8.6%. The highlights of our strategy involved an E-selective olefin cross-metathesis, Suzuki cross-coupling, and a copper(I)-catalyzed coupling reaction.

The synthetic tubulysin derivative, tubugi-1, improves the innate immune response by macrophage polarization in addition to its direct cytotoxic effects in a murine melanoma model.

Synthetic tubugis are equally potent but more stable than their natural forms. Their anticancer potential was estimated on a solid melanoma in vitro and in vivo. Tubugi-1 induced the apoptosis in B16 cells accompanied with strong intracellular production of reactive species, subsequently imposing glutathione and thiol group depletion. Paradoxically, membrane lipids were excluded from the cascade of intracellular oxidation, according to malondialdehyde decrease. Although morphologically apoptosis was typical, externalization of phosphatidylserine (PS) as an early apoptotic event was not detected. Even their exposition is pivotal for apoptotic cell eradication, primary macrophages successfully eliminated PS-deficient tubugi-1 induced apoptotic cells. The tumor volume in animals exposed to the drug in therapeutic mode was reduced in comparison to control as well as to paclitaxel-treated animals. Importantly, macrophages isolated from tubugi-1 treated animals possessed conserved phagocytic activity and were functionally and phenotypically recognized as M1. The cytotoxic effect of tubugi-1 is accomplished through its ability to polarize the macrophages toward M1, probably by PS independent apoptotic cell engulfment. The unique potential of tubugi-1 to prime the innate immune response through the induction of a specific pattern of tumor cell apoptosis can be of extraordinary importance from fundamental and applicable aspects.

Prostate-Specific Membrane Antigen-Specific Antitumor Activity of a Self-Immolative Tubulysin Conjugate.

Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and it is also present on the neovasculature within many non-prostate solid tumors. Herein, we report on the construction and biological testing of novel tubulysin B-containing therapeutic agents for the treatment of PSMA-expressing cancer. One of these compounds, EC1169, emerged as a lead candidate for preclinical development and phase 1 clinical testing. This water-soluble conjugate was shown to have high affinity for PSMA-positive cells. When tested in vitro, EC1169 was found to inhibit the growth of PSMA-positive cells, but it displayed no activity against PSMA-negative cells. Brief treatment of nude mice bearing PSMA-positive LNCaP human xenografts with EC1169 led to complete remissions and cures. Furthermore, this activity occurred in the absence of weight loss. In contrast, the nontargeted tubulysin B drug proved to be inactive against the LNCaP tumor model when administered at doses near to or greater than the maximum tolerated level. PSMA-negative KB tumors did not appreciably respond to EC1169 therapy, thereby confirming this compound's targeted specificity for PSMA-positive cells. Finally, treatment of LNCaP-bearing mice with docetaxel (the most active chemotherapeutic agent approved for late stage prostate cancer therapy) was found to produce only modest anti-tumor activity, and this outcome was also associated with severe weight loss. Taken together, these results strongly indicate that PSMA-positive tumors may be effectively treated using highly potent, PSMA-targeted small-molecule drug conjugates using regimens that do not cause undesirable side effects.

Chlorosulfonic Acid Supported Piperidine-4-carboxylic Acid (PPCA) Functionalized Fe3O4 Nanoparticles (Fe3O4-PPCA): The Efficient, Green and Reusable Nanocatalyst for the Synthesis of Pyrazolyl Coumarin Derivatives under Solvent-Free Conditions.

BACKGROUND: In this study, we developed a convenient methodology for the synthesis of coumarin linked to pyrazolines and pyrano [2,3-h] coumarins linked to 3-(1,5-diphenyl-4,5- dihydro-1H-pyrazol-3-yl)-chromen-2-one derivatives using Chlorosulfonic acid supported Piperidine-4-carboxylic acid (PPCA) functionalized Fe3O4 nanoparticles (Fe3O4-PPCA) catalyst. MATERIALS AND METHODS: Fe3O4-PPCA was investigated as an efficient and magnetically recoverable Nanocatalyst for the one-pot synthesis of substituted coumarins from the reaction of coumarin with a variety of aromatic aldehydes in high to excellent yield at room temperature under solvent-free conditions. The magnetic nanocatalyst can be easily recovered by applying an external magnet device and reused for at least 10 reaction runs without considerable loss of reactivity. RESULTS AND CONCLUSION: The advantages of this protocol are the use of commercially available materials, simple and an inexpensive procedure, easy separation, and an eco-friendly procedure, and it shows good reaction times, good to high yields, inexpensive and practicability procedure, and high efficiency.

Immobilizing argatroban and mPEG-NH2 on a polyethersulfone membrane surface to prepare an effective nonthrombogenic biointerface.

Systemic anticoagulation is not suitable for hemodialysis (HD) patients with a high risk of bleeding in the clinic. An HD membrane that provides a localized anticoagulation membrane surface may be a promising strategy to solve this intractable problem for HD patients. Herein, we modified a nonthrombogenic polyethersulfone (PES) dialyzer membrane by grafting argatroban (AG) and methoxy polyethylene glycol amine (mPEG-NH2) via a polydopamine (PDA) strategy. The PES substrates were immersed in an alkaline dopamine solution for 24 h, and then, AG and mPEG-NH2 were sequentially grafted covalently onto the resultant membrane. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) were utilized to confirm the successful introduction of PDA and the immobilization of AG and mPEG-NH2. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to observe the surface structure and morphology after the surface modification. The excellent antithrombotic abilities of the modified membrane were demonstrated by the suppression of platelet adhesion and activation, prolongation of clotting times, and inhibition of thrombin generation and complement activation. This work describes an efficient and convenient method to immobilize AG and mPEG-NH2 to create a nonthrombogenic biointerface for blood-contacting devices such as HD membranes.

Pipecolic esters as minimized templates for proteasome inhibition.

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.

Development of a stent capable of the controlled release of basic fibroblast growth factor and argatroban to treat cerebral aneurysms: In vitro experiment and evaluation in a rabbit aneurysm model.

An ideal stent to treat cerebral aneurysms should have an antithrombotic effect on the inner stent blood-facing side and a tissue organization effect on the outer aneurysmal side of the stent. The objective of this study is to evaluate the feasibility of a drug containing stent in the in vivo treatment of cerebral aneurysms. Argatroban, an antithrombotic drug, is encapsulated in biodegradable poly (d,l-lactide-co-glycolide) (PLGA) microspheres for the controlled release with an in vitro study conducted to evaluate the drug release and anticoagulation behavior of released drug. Basic fibroblast growth factor (bFGF), an organization drug, is released from gelatin hydrogels. The stents are coated with gelatin hydrogels incorporating bFGF and PLGA microspheres containing argatroban, and applied to the carotid artery aneurysm of an elastase-induced rabbit model. Most of the aneurysm cavity is occupied by loose connective tissues in the group treated with drug-coated stents, whereas extensive massive hematomas are observed in the group treated with drug-free stents. The occurrence rate of in-stent thrombus is small in the drug-coated stents. The stent incorporating bFGF and PLGA microspheres containing argatroban is an effective device for cerebral aneurysm treatment. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2185-2194, 2019.

Elucidating the multiple structures of pipecolic acid by rotational spectroscopy.

The complex conformational space of the non-proteinogenic cyclic amino acid pipecolic acid has been explored in the gas phase for the first time. Solid pipecolic acid samples were vaporized by laser ablation and expanded in a supersonic jet where the rotational spectral signatures owing to nine different conformers were observed by Fourier transform microwave spectroscopy. All species were identified by comparison of the experimental rotational and nuclear quadrupole coupling constants with those predicted theoretically. Observation of type-III conformers, leading to a difference when compared against the conformational behavior of the analog amino acid proline, has been interpreted by an increment in steric hindrance when increasing the number of carbons present in the ring.

A More Sustainable Process for Preparation of the Muscarinic Acetylcholine Antagonist Umeclidinium Bromide.

A more sustainable process for the synthesis of the long-acting muscarinic acetylcholine antagonist umeclidinium bromide is described. Specifically, we report the synthesis of ethyl 1-(2-chloroethyl)-4-piperidinecarboxylate, a key intermediate in the preparation of umeclidinium bromide, in good yields using triethylamine, as well as the identification and characterization of the by-product formed in this reaction. This new method of synthesis leads to an improvement in yield over that of previously reported protocols using potassium carbonate as base (65.6 % versus 38.6 %). Moreover, in the final synthetic step of the process to obtain umeclidinium bromide, we were able to replace the use of toxic solvents (acetonitrile/chloroform) with water. The use of this green solvent allowed precipitation of the active pharmaceutical ingredient (API) from the reaction medium with high purity and in high yield. Overall, we have developed a more efficient and environmentally friendly process for the synthesis of the umeclidinium bromide API with a higher overall yield (37.8 % versus previously reported overall yield of 9.7 %).

Structure, Total Synthesis, and Biosynthesis of Chloromyxamides: Myxobacterial Tetrapeptides Featuring an Uncommon 6-Chloromethyl-5-methoxypipecolic Acid Building Block.

Soil-living microbes are an important resource for the discovery of new natural products featuring great structural diversity that are reflective of the underlying biosynthetic pathways as well as incorporating a wide range of intriguing small-molecule building blocks. We report here the full structural elucidation, total synthesis, and biosynthesis of chloromyxamides, a new class of tetrapeptides that display an unprecedented 6-chloromethyl-5-methoxypipecolic acid (CMPA) substructure. Chemical synthesis-including an approach to access the CMPA unit-was pursued to confirm the structure of the chloromyxamides and enabled determination of the absolute configuration in the CMPA ring. A model for the nonribosomal assembly of chloromyxamides was devised on the basis of the combined evaluation of the biosynthetic gene cluster sequence and the feeding of stable isotope-labeled precursors. This provided insight into the formation of the various chloromyxamide derivatives and the biogenesis of the CMPA unit.

Pre-clinical evaluation of EC1456, a folate-tubulysin anti-cancer therapeutic.

EC1456 is a folate-tubulysin conjugate constructed with an all-D enantiomeric spacer/linker configuration. When tested against folate receptor (FR)-positive cells, EC1456 demonstrated dose-responsive activity with an approximate 1000-fold level of specificity. Treatment of nude mice bearing FR-positive human xenografts (as large as 800 mm3) with non-toxic doses of EC1456 led to cures in 100% of the mice. Combinations of low dose EC1456 with standard of care agents such as platins, taxanes, topotecan and bevacizumab, safely and significantly augmented the growth inhibitory effects of these commonly used agents. When tested against FR-positive human tumor xenograft models having confirmed resistance to a folate-vinca alkaloid (vintafolide), cisplatin or paclitaxel, EC1456 was found to generate partial to curative responses. Taken together, these studies demonstrate that EC1456 has significant anti-proliferative activity against FR-positive tumors, including models which were anticancer drug resistant, thereby justifying a Phase 1 trial of this agent for the treatment of advanced human cancers.

Argatroban is stable in citrated whole blood for 24 hours.

INTRODUCTION: Argatroban is a direct thrombin inhibitor used as an anticoagulant for patients who have Heparin induced thrombocytopenia. Quantification can be performed using a dilute thrombin time or anti-iia assay. Our preferred method is Hemoclot Thrombin Inhibitor Assay (HTI).To the best of our knowledge, no one has published on the stability of plasma argatroban in whole citrated blood at room temperature. METHODS: Forty matched samples obtained from 4 patients receiving argatroban. 1 mL of the whole blood was removed from the sample into a labelled plastic tube and left on the laboratory bench at room temperature for 24 hours prior to centrifugation. The remaining sample was spun at 2000 g for 10 minutes and the plasma aliquoted off and labelled 0 hour and stored at -80°C prior to testing. At 24 hours the plastic tube containing whole blood was centrifuged at 2000 g for 10 minutes and the plasma aliquoted off and labelled 24 hours and stored at -80°C prior to testing. HTI assay was used for plasma argatroban determination. RESULTS: The lowest argatroban level obtained was 0.16 µg/mL at 0 hour; 0.18 µg/mL at 24 hours from the same pair, the highest argatroban level obtained was 1.72 µg/mL at 0 hour;1.76 µg/mL for the same pair at 24 hours. The mean result for 0 hour was 0.57 µg/mL and 0.60 µg/mL at 24 hours. CONCLUSION: This study proposes that patients receiving treatment in hospitals who cannot provide a dedicated argatroban plasma concentration method could have their samples sent on whole blood within 24 hours of venepuncture to a laboratory who could provide the test.

Synthesis and biocompatibility of an argatroban-modified polysulfone membrane that directly inhibits thrombosis.

Anticoagulation therapy plays a vital role in the prevention of blood clot formation during hemodialysis and hemofiltration, especially for critical care patients. Here, we synthesized a novel argatroban (Arg)-modified polysulfone (PSf) membrane for anticoagulation. Arg was grafted onto the PSF membrane via chemical modification to increase membrane hydrophilicity. Protein adsorption, coagulation, as well as activation of platelets and complement systems were greatly reduced on the Arg-modified PSf membrane. Thus, the recalcification time and the activated partial thrombin time (APTT) were increased after the modification. In comparison with the pristine PSf membrane, the Arg-modified PSf membrane showed better hemocompatibility and anticoagulation properties, indicating its potential for applications in hemodialysis and hemofiltration. Modification of the PSf membrane has been investigated in attempts to further enhance the anticoagulation properties of the hemodialysis membranes, including a heparin-modified PSf membrane. However, heparin can inhibit plasma-free thrombin, and cause the occurrence of heparin-induced thrombocytopenia (HIT), which increases the risk of bleeding during dialysis in critical care patients. To address this problem, we modified PSf membrane with as a novel direct thrombin inhibitors, argatroban (Arg). It can reversibly bind to thrombin, inhibiting not only the plasma-free thrombin in the blood, but also clot-bound thrombin.

An economically and environmentally acceptable synthesis of chiral drug intermediate L-pipecolic acid from biomass-derived lysine via artificially engineered microbes.

Deficiency in petroleum resources and increasing environmental concerns have pushed a bio-based economy to be built, employing a highly reproducible, metal contaminant free, sustainable and green biomanufacturing method. Here, a chiral drug intermediate L-pipecolic acid has been synthesized from biomass-derived lysine. This artificial bioconversion system involves the coexpression of four functional genes, which encode L-lysine α-oxidase from Scomber japonicus, glucose dehydrogenase from Bacillus subtilis, Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida, and lysine permease from Escherichia coli. Besides, a lysine degradation enzyme has been knocked out to strengthen the process in this microbe. The overexpression of LysP improved the L-pipecolic acid titer about 1.6-folds compared to the control. This engineered microbial factory showed the highest L-pipecolic acid production of 46.7 g/L reported to date and a higher productivity of 2.41 g/L h and a yield of 0.89 g/g. This biotechnological L-pipecolic acid production is a simple, economic, and green technology to replace the presently used chemical synthesis.